Granulocyte-angiotensin system. Identification of angiotensinogen as the plasma protein substrate of leukocyte cathepsin G

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Abstract
Cathepsin G, a human lysosomal neutral protease, converts angiotensin I to angiotensin II and cleaves angiotensin II from a plasma protein substrate. Experiments were designed that identified and characterized cathepsin G substrate as human angiotensinogen. A total of 2, 5 and 10 .mu.g of purified substrate, incubated with 2 .mu.l of partially purified human renin (2 Goldblatt U/mg) for 60 min at 37.degree. C, generated 2, 9 and 22 pmol of angiotensin I. Cathepsin G substrate and renin substrate activities copurified during Affi-Gel Blue affinity chromatography, hydroxylapatite chromatography, phenyl-Sepharose chromatography and S-200 gel filtration. Disc gel electrophoresis of 10 .mu.g of purified protein gave a single band containing both activities. The amino-terminal sequence contained the covalent structure of angiotensin I and was Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-X-Glu-Ser-Thr-Cys-Glu-. Reduced and unreduced angiotensinogens were subjected to sodium dodecyl sulfate gel electrophoresis, and each gel showed 2 bands of MW 65,000 and 62,000. The isoelectric point of the MW 65,000 form was pH 4.5-4.3 and the MW 62,000 form was pH 4.9. Functional, structural and physiochemical evidence demonstrates that the substrate of cathepsin G is angiotensinogen. Human neutrophils may utilize angiotension I or angiotensinogen as substrate for angiotensin II generation. The granulocyte-angiotensin system does not require renin or converting enzyme and may function as a mobile effector pathway which modulates tissue blood flow and/or vascular permeability.